1. Field of the Invention
The present invention relates to a process for preparing haptens for immunoassay of phosphorothioate pesticides, more specifically, to a process for preparing haptens for immunoassay of organophosphorus phosphorothioate pesticides, by reacting O-methyl(ethyl) dichlorothiophosphate with a phenolic compound to obtain O-methyl(ethyl) O-aryl chlorothiophosphate, and reacting O-methyl(ethyl) O-aryl chlorothiophosphate thus obtained with aminocarboxylic acid.
2. Background of the Invention
Since Schrader's discovery of an organophosphorus compound possessing insecticidal activity in the 1930's, lots of organophosphorus pesticides with high biological activities have been actively developed in the art, in line with the movement of prohibiting the use of organochlorine pesticides. Nowadays, organophosphorus pesticides hold a large majority in current pesticides. Further, it is remarkable that about 100,000 of them have been identified to possess insecticidal activity, and more than 100 of them are commercially available.
Organophosphorus pesticides are classified into phosphate, phosphorothioate, phosphorothiolate, phosphorodithioate, phosphonate, phosphonothioate, phosphonodithioate, phosphorothiolothinate, and phosphoroamidate, depending on their chemical structures around a phosphorus atom (see: Table 1). Among them, phosphorothioate and phosphorodithioate are considered to be the most important pesticides in agriculture and have become the main subject of assaying residual pesticides.
The assay of residual pesticide has been carried out mainly by the aid of GC or HPLC, both of which have innate disadvantages that a time-consuming step of pre-treatment, high-priced machinery and tools, and labors with professional techniques are required and, in the case of GC, it is impossible to assay thermolabile material and in the case of HPLC, it is hard to assay pesticides having no chromophore. To solve these problems, many attempts to employ an immunoassay method for analysing residual pesticides, which was mainly used for assay of bio-components or a clinical diagnosis, began to be made in the 1970's. Immunoassay of residual pesticides is more favorable than the conventional methods in the points that: it is highly sensitive; pre-treatment of samples is not required; and, it costs a less deal since it allows the rapid assay of multiple samples in a simultaneous manner.
Immunoassay is based on the specific binding with a high affinity between antibodies and antigens. Thus, to develop immunoassay, an appropriate antigen should be prepared to generate antibodies against a substance to be assayed. However, low-molecular weight materials, such as pesticides, cannot serve as antigens in themselves and thus, antibodies cannot be generated. Under the circumstances, the need has been raised that haptens having a similar structure to pesticide and a functional groups capable of forming covalent bonds with proteins, should be synthesized for preparing pesticide-specific antigens. Also, synthesis of haptens has been required for preparing an enzyme tracer and a coating antigen, a competitor to be used for a competitive-immunoassay method.
TABLE 1Classification of organophosphorus pesticidesClassStructureExamplePhosphateDichlorvos PhosphorothioateParathion PhosphorothiolateOmethoate PhosphorodithioateMalathion Phosphonatetrichlorfon PhosphonothioateEPN PhosphonodithioateFonfos PhosphorothiolothionateEthoprop PhosphoroamidateDimefox
In general, haptens used for immunoassay of phosphorothioate pesticides have a chemical structure as following:
wherein,                R1 is methyl or ethyl group;        R2 is aryl group;        R3 is hydrogen or alkyl group; and,        R4 is alkylidene group.        
Among the above haptens, haptens having the structure in which R3 is hydrogen and R4 is di- or pentamethylene group have been synthesized so far. The haptens having the structure in which R3 is hydrogen and R4 is dimethylene group have been synthesized by a process comprising the steps of: (i) reacting 3-aminopropanoic acid (1) with benzylchloroformate to obtain 3-(benzyloxycarbonylamino)propanoic acid (2); (ii) reacting 3-(benzyloxycarbonylamino)propanoic acid (2) thus obtained with tert-butanol in the presence of dicyclohexalcarbodiimide(DCC) to obtain tert-butyl 3-(benzyloxycarbonylamino)propanoate (3); (iii) eliminating amino protective group from tert-butyl 3-(benzyloxycarbonylamino)propanoate (3) thus obtained by way of hydrogenation using a catalyst to obtain tert-butyl-3-aminopropanoate (4); (iv) reacting tert-butyl 3-aminopropanoate (4) thus obtained with O-methyl dichlorothiophosphate (5) to obtain tert-butyl 3-[chloro(methoxyl)phosphorothioylamino]propanoate (6); (v) reacting tert-butyl 3-[chloro(methoxyl) phosphorothioylamino]propanoate thus obtained with a sodium salt of phenol (7) to obtain tert-butyl 3-[methoxy(aryloxy)phosphorothioylamino]propanoate (8); and, (vi) removing tert-butyl protective group from tert-butyl 3-[methoxy(aryloxy)phosphorothioylamino]propanoate (8) with an aid of trifluoroacetic acid(TFA) to obtain 3-[methoxy(aryloxy)phosphorothioylamino]propanoic acid (9).

It has been known that O-alkyl O-aryl N-(2-carboxyalkyl)phosphoramidothioates synthesized by the above process is can be a haptens highly preferable for generating antibodies for phosphothioate pesticides. However, the said process has revealed shortcomings that it is very complicated, time- and cost-consuming and poorly yielded, since the said method goes through a process comprising total six steps or seven steps provided a step of preparing a sodium salt of phenol is added.
Under the circumstances, there are strong reasons for developing a process for preparing haptens for immunoassay of phosphothioate pesticide in a more efficient manner.